PyroMark CpG Assays

Predesigned assays for quantification of CpG methylation by Pyrosequencing

Advanced assay design algorithms provide optimized results

Easy-to-order assays for gene-specific or methylation array CpG sites

Comprehensive coverage of CpG sites in human, mouse, and rat genomes

Convenient download of run files and analysis settings

Top performance from a cost-effective solution

PyroMark CpG Assays are the only genomewide, predesigned methylation assays for Pyrosequencing analysis that use a tailored design algorithm for highly specific assay design and successful CpG methylation results. Our upgraded assay database now includes over 84,000 individual assays for gene-specific human, mouse, and rat CpG sites. We now also offer predesigned assays for validating results from methylation arrays, such as the Infinium HumanMethylation450K and MethylationEPIC BeadChip arrays.

Search in GeneGlobe

You can search for the following terms:

Entrez Gene IDs (e.g., 835)

RefSeq IDs (e.g., NM_032983, NP_116765)

Gene symbols (e.g., CASP2)

Cat. no. (e.g., SI00299551, QT01342509)

Sanger ID or Accession (e.g., hsa-let-7b, MI0000063)

CpG loci identification numbers (CG#) (e.g., CG17753661)

Do not enter species information in the Search box. Use the drop-down list to select your species of interest.
When searching for miRNAs, do not omit the hyphens. Use hyphens or spaces (e.g., search for hsa-let-7b or hsa let 7b. Do not search for hsalet7b).

[A] The GeneGlobe interface for PyroMark CpG Assays affords access to multiple targets in CpG islands, which allows the user to pick and choose the particular regions in a CpG island to analyze. [B] Pyrosequencing data show the analysis of two different positions within the CpG island associated with the gene ESR1.|The PCR performance of PyroMark CpG Assays for 310 randomly selected genes was evaluated. A total of 305 assays (98%) resulted in the predicted PCR amplicon, usable as template for subsequent Pyrosequencing.|The Pyrosequencing performance of PyroMark CpG Assays for 310 randomly selected genes was evaluated. A total of 305 assays (98%) resulted in the predicted PCR amplicon, usable as template for subsequent Pyrosequencing. From these assays, 281 resulted in successful Pyrosequencing results. Of these successful assays, 229 (75.1%) required no further review by the user, and 52 (17%) were successful after user review. Only 24 (7.9%) evaluated assays failed.|The GeneGlobe interface for searching and ordering PyroMark CpG Assays provides access to multiple targets in CpG islands, allowing users to pick and choose the exact regions in a CpG island for analysis. By combining different assays, users can quantify methylation of as many target sites as needed, whether these are consecutive or not.|

Primer design optimized for high performance

Highly specific and unbiased amplification is especially important for methylation analysis by Pyrosequencing. Because bisulfite converted DNA is a less complex template, it can be challenging to amplify specifically and at high yields, but with predesigned PyroMark CpG Assays, this is not a problem. The PCR and sequencing primers included in PyroMark CpG Assays have been checked against the entire human, mouse, or rat bisulfitome and are delivered at optimal concentrations for high performance. Together with the unique DNA protect technology of EpiTect Fast Bisulfite Conversion Kits and the carefully formulated reagents of the PyroMark PCR Kit, these predesigned assays ensure high yield and quality of sequencing template and increase reliability of downstream Pyrosequencing analyses.

Assays for analyzing gene-specific CpG sites

PyroMark CpG Assays enable methylation analysis of specific targets across the genome. Our database now contains over 84,000 predesigned assays covering more than 80% of gene-specific CpG islands for the human, mouse, and rat genomes. These assays are designed using a carefully tailored algorithm that generates optimal assay design based on bisulfite converted DNA. As such, these fast and easy-to-order assays minimize time spent on assay optimization and maximize analysis success. PyroMark CpG Assays expand the streamlined Pyrosequencing workflow to enable methylation analysis of virtually any CpG island in the human, mouse, or rat genome (see figure Analysis of ESR1).

Assays for methylation array validation

Hybridization arrays enable high-throughput discovery of variations in DNA methylation, but the results must be experimentally verified to confirm that the correct variations are identified. Pyrosequencing is a cost-effective technique highly suited for this purpose, providing quantitative analysis of short- to medium-length sequences with high accuracy. We now offer predesigned PyroMark CpG Assays for validating results from HumanMethylation450K and MethylationEPIC BeadChip experiments. These new predesigned assays for methylation array validation offer a convenient way to verify experimental results, and determine exact methylation levels of CpG sites of interest, without the need to manually design a separate assay for each individual target of interest. Our dedicated search page lets users simply enter the CpG loci identification numbers (CG#) of interest to easily search our assay database and order ready-to-use PyroMark CpG Assays.

The advantages of GeneGlobe

QIAGEN's GeneGlobe Web portal is a one-stop online interface that grants access to our extensive range of assay technologies in a gene- or pathway-specific context. GeneGlobe brings together state-of-the-art search and design algorithms, up-to-date sequence data, and our innovative technologies to facilitate discovery. Simply enter a search term for the gene or pathway of interest, and let GeneGlobe show you how to advance your research in epigenetics, or any other area of interest (see figure Ready-to-use methylation assays).

Procedure

PyroMark CpG Assays include PCR and sequencing primers for quantification of DNA methylation by Pyrosequencing. A biotinylated PCR product is generated from the bisulfite converted DNA template and is bound to streptavidin-coated Sepharose beads. The beads are captured with the vacuum tool on the PyroMark Vacuum Workstation, where they are thoroughly washed and subsequently denatured, generating single-stranded DNA suitable for Pyrosequencing. This template DNA is released into the Pyrosequencing reaction plate containing the sequencing primer provided with PyroMark CpG Assays. After primer annealing, the plate is placed into the PyroMark instrument, and the sequencing reaction is started.

Applications

PyroMark CpG Assays enable highly accurate, quantitative methylation analysis of gene-specific human, mouse, and rat CpG sites as well as targets from the HumanMethylation450K and the MethylationEPIC BeadChip for epigenetics research.